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The EtOAc extract of licorice (Glycyrrhiza uralensis roots) exhibited considerable PPAR-gamma ligand-binding activity. Bioassay-guided fractionation of the extract using a GAL-4-PPAR-gamma chimera assay method resulted in the isolation of two isoflavenes, one of which is a new compound named dehydroglyasperin D, an isoflavan, two 3-arylcoumarins, and an isoflavanone as the PPAR-gamma ligand-binding active ingredients of licorice. The isoprenyl group at C-6 and the C-2' hydroxyl group in the aromatic ring-C part in the isoflavan, isoflavene, or arylcoumarin skeleton were found to be the structural requirements for PPAR-gamma ligand-binding activity. Glycyrin, one of the main PPAR-gamma ligands of licorice, significantly decreased the blood glucose levels of genetically diabetic KK-A(y) mice.

The metabolic syndrome, including type 2 diabetes, insulin resistance, obesity/abdominal obesity, hypertension and dyslipidemia, is a major public health problem. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands such as thiazolidinediones are effective against this syndrome. In this study, we showed that nonaqueous fractions of licorice (Glycyrrhiza uralensis Fisher) extracted with ethanol, ethyl acetate and acetone, but not an aqueous extract, had PPAR-gamma ligand-binding activity with a GAL4-PPAR-gamma chimera assay. Some prenylflavonoids including glycycoumarin, glycyrin, dehydroglyasperin C and dehydroglyasperin D, a newly found compound, were identified as active compounds with PPAR-gamma ligand-binding activity in the nonaqueous fraction of licorice. A licorice ethanolic extract contained these four active compounds at a total concentration of 16.7 g/100 g extract. Feeding the licorice ethanolic extract at 0.1-0.3 g/100 g diet [approximately 100 to 300 mg/(kg body x d)] for 4 wk decreased (P < 0.05) blood glucose level in younger (6 wk old) and older (13 wk old) diabetic KK-Ay mice and reduced (P < 0.05) weights of intra-abdominal adipose tissues in high fat diet-induced obese C57BL mice. An increase in blood pressure in spontaneously hypertensive rats was suppressed (P < 0.01) by 3 wk of oral administration of the licorice ethanolic extract at 300 mg/(kg body x d). These findings indicate that licorice ethanolic extract is effective in preventing and ameliorating diabetes, ameliorating abdominal obesity and preventing hypertension, and suggest that licorice ethanolic extract would be effective in preventing and/or ameliorating the metabolic syndrome.

Freeze-dried tendon allograft sterilized with gamma irradiation could be a reasonable option for ligament substitute. In the current study, the effects of freezing or freeze-drying followed by gamma irradiation on remodeling were analyzed biomechanically in a rat patellar tendon transplantation model at the time of harvest and during a 24-week healing period. The grafts were divided into four groups: fresh-freezing, freeze-drying, fresh-freezing followed by gamma irradiation, and freeze-drying followed by gamma irradiation. Before transplantation, the fresh-frozen grafts and the freeze-dried grafts showed significantly greater tensile strength than the gamma-irradiated grafts. However, at 4 weeks, the tensile strength of each group decreased to the equivalent level, which was approximately 20% of the tensile strength at Time 0, then increased gradually with time. At 24 weeks, the mean tensile strength of each transplanted graft achieved as much as 50% of the tensile strength at Time 0. The change in the tangent modulus with time followed a similar pattern as changes in the tensile strength. This study assumed that the extraarticular tendon transfer model was suitable for evaluating anterior cruciate ligament graft healing. These data suggest that the freeze-dried tendon allografts sterilized by gamma radiation could be a suitable substitute for anterior cruciate ligament reconstruction, if care is taken to protect the graft during the early stage after transplantation (< 4 weeks).

Immunocytochemical electron-microscopic observation indicated that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) and/or its degradation products are localized in small spherical bodies having a diameter of 0.4-1.2 micro m in naturally senescing leaves of wheat (Triticum aestivum L.). These Rubisco-containing bodies (RCBs) were found in the cytoplasm and in the vacuole. RCBs contained another stromal protein, chloroplastic glutamine synthetase, but not thylakoid proteins. Ultrastructural analysis suggested that RCBs had double membranes, which seemed to be derived from the chloroplast envelope, and that RCBs were further surrounded by the other membrane structures in the cytoplasm. The appearance of RCBs was the most remarkable when the amount of Rubisco started to decrease at the early phase of leaf senescence. These results suggest that RCBs might be involved in the degradation process of Rubisco outside of chloroplasts during leaf senescence.

The N-use efficiency for photosynthesis was higher in a C(4) plant, maize, than in a C(3) plant, rice, including rbcS antisense rice with optimal ribulose-1,5-bisphosphate carboxylase (Rubisco) content for CO(2)-saturated photosynthesis, even when photosynthesis was measured under saturating CO(2) conditions. The N cost for the C(4) cycle enzymes in maize was not large, and the lower amount of Rubisco allowed a greater N investment in the thylakoid components. This greater content of the thylakoid components as well as the CO(2) concentrating mechanism may support higher N-use efficiency for photosynthesis in maize.

Low irradiance in the early phase of grain filling in rice often results in a low grain yield, but its effects on the partitioning of previously or recently assimilated carbon within the plant or panicle have not been seriously examined. The objective of this study was to demonstrate the effect of shading during the different stages in the early phase of grain filling on the partitioning of previously or recently assimilated carbon among constituent organs and into superior and inferior spikelets of the panicle in rice (Oryza sativa L. 'Sasanishiki') plants using 13C as a tracer. Plants were grown either under low (shading) or moderate (non-shading) irradiance (120 and 800 micromol quantum m(-2) s(-1)) for 3 or 4 d before or after the 13CO2 feeding at heading, full-heading or milky stages during the early phase of grain filling. Four days after the 13CO2 feeding, the proportion of labelled (previously assimilated) carbon partitioned into the panicle was 17% higher in plants grown under low irradiance compared with plants grown under moderate irradiance at the full-heading stage (7-11 d after heading), while the proportion partitioned into the culm was 13% lower. The light treatments for 3 d were conducted before the 13CO2 feeding and partitioning of the labelled (recently-assimilated) carbon into spikelets was examined 6 h after feeding. The amount of labelled carbon partitioned into the spikelets of the secondary branch (inferior grains) in the plants grown under low irradiance was only 31% when compared with plants grown under moderate irradiance at the full-heading stage, although the partitioning of labelled carbon into the apical spikelets of the primary branch (superior grains) was not affected by the light treatments. These results clearly indicate that preferential partitioning of assimilated carbon into the panicle occurs under low irradiance at around 7-11 d after heading and that the priority of superior spikelets for assimilated carbon intensifies. This phenomenon is thought to be an important strategy for such rice cultivars as used in this study to achieve a certain proportion of ripened grains even under light limited conditions.

While techniques to arthroscopically fix a large fragment (3 cm or more in width) of an avulsion fracture of the posterior cruciate ligament with multiple cannulated screws have been described, those for a smaller fragment (1.5 cm or less in width) have not been well documented. We present 2 techniques to arthroscopically manage the small fragment with a single cannulated screw or with sutures and a pullout button. The technique makes it possible to achieve bony union by 4 months following surgery and to allow early and easy screw removal leading to a more complete union.

Lysates of chloroplasts isolated from naturally senescing wheat leaves were incubated in darkness. The 44-kDa fragment, lacking the N-terminal-side portion of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (LSU), was found by immunoblotting with the LSU site-specific antibodies. Analysis of its N-terminal amino acid sequence indicated that the LSU was specifically cleaved at the peptide bond between Phe-40 and Arg-41. The site was located on the surface of the molecule and faced outward. Such cleavage of the LSU has not been previously reported. It is indicated that the cleavage was triggered by an unknown protease existing in chloroplasts.

In order to determine whether there is a difference in effect on cell morphology and function between two common arthroscopic irrigation solutions, primary cultures of cells derived from the surgically excised human menisci were incubated for 3 or 6 h in lactated Ringer's solution, isotonic sodium chloride solution, or serum-free cell culture medium (negative-control condition). Cell integrity was blindly evaluated by three independent examiners scoring photomicrographs of the cell cultures on a battery of five-point scales for abnormality of cell shape, irregularity of cell membrane, change of cell size and cell density. Cell cultures were also quantitatively assayed by semi-quantitative reverse-transcription-polymerase-chain-reaction for mRNA of alpha1 (I) procollagen, alpha1 (II) procollagen, aggrecan and heat-shock protein 70 to assess functional consequences of exposure to the solutions. There was a statistically significant difference in cell integrity scores between either lactated Ringer's solution or serum-free cell-culture medium and isotonic sodium chloride solution with greater damage to cells displayed. Scores for lactated Ringer's solution did not differ from those for serum-free cell-culture medium. There were no significant differences in mRNA expression level among the treatment conditions. It was concluded that the lactated Ringer's solution better maintained human meniscus cell integrity than the isotonic saline.

Purpose: To biomechanically evaluate a new fixation device, DSP (Double Spike Plate; Meira Corp, Nagoya, Aichi, Japan), for pullout graft fixation., Type of Study: Biomechanical study., Methods: A porcine tibia in which 8-mm diameter drill holes had been made from the medial tibial metaphysis to the anterior cruciate ligament attachment was rigidly fixed to a tension analyzer. A quadrupled graft consisting of 2 double-looped bovine tendons was prepared with No. 3 polyester sutures placed distally. The graft was passed through the drill hole, and its proximal loop ends were rigidly fixed to a load cell for monitoring graft tension. The graft's distal ends were connected to the DSP by tying the sutures to the top hole in the DSP. The graft tension was predetermined at 49 N (n = 5) or 98 N (n = 5). This tension was maintained for 5 minutes with a suture passed through the bottom hole of the DSP. The plate was fixed to the tibia by hammering its spikes into the bone under the index tension. Finally, the fixation was completed by inserting a screw., Results: Although the graft tension immediately increased to 69 +/- 11 N or 133 +/- 14 N during hammering, it gradually reduced to 49 +/- 10 N or 100 +/- 7 N 5 minutes later., Conclusions: This study shows that graft fixation under a predetermined tension can be achieved with the DSP.

Background: Spontaneous crescentic glomerulonephritis (SCG)/Kj mice are a candidate for suitable animal model of human pauci-immune crescentic glomerulonephritis (GN). In the present study, we used renal biopsy technique and analyzed time sequence correlations among crescent formation and glomerular neutrophil infiltration in SCG/Kj mice., Methods: We observed the progress of GN in SCG/Kj mice according to the urinary abnormalities, and performed the serial renal biopsies. The kinetics of histopathology and glomerular neutrophil influx corresponding disease stage were examined by enzyme-histochemistry and immunohistochemistry., Results: We divided natural course of GN into three periods in view of urinalysis: a proteinuria-negative/hematuria-negative (P- H-) period, followed by a proteinuria-positive/hematuria-negative (P+ H-) period, and finally a proteinuria-positive/hematuria-positive (P+ H+) period. Endocapillary proliferation phase existed in P+ H- period, whereas crescent formation occurred and extended in P+ H+ period. In P+ H- period, prominent glomerular neutrophil infiltration was observed, while these numbers decreased with the progression of crescent formation., Conclusion: These observations suggest that there is a good correlation between urinalysis and histopathological events of SCG/Kj mice, and that endocapillary proliferation, which contains neutrophil infiltration, may contribute to the subsequent crescent formation in these mice.

Previous work has demonstrated that the large subunit (rbcL) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) from wheat is cleaved at Gly-329 by the Fe(2+)/ascorbate/H(2)O(2) system (Ishida, H., Makino, A., and Mae, T. (1999) J. Biol. Chem. 274, 5222-5226). In this study, we found that the rbcL could also be cleaved into several other fragments by increasing the incubation time or the Fe(2+) concentration. By combining immunoblotting with N-terminal amino acid sequencing, cleavage sites were identified at Gly-404, Gly-380, Gly-329, Ala-296, Asp-203, and Gly-122. Conformational analysis demonstrated that five of them are located in the alpha/beta-barrel, whereas Gly-122 is in the N-terminal domain but near the bound metal in the adjacent rbcL. All of these residues are at or very close to the active site and are just around the metal-binding site within a radius of 12 A. Furthermore, their C(alpha)H groups are completely or partially exposed to the bound metal. A radical scavenger, activation of RuBisCo, or binding of a reaction-intermediate analogue to the activated RuBisCo, inhibited the fragmentation. These results strongly suggest that the rbcL is cleaved by reactive oxygen species generated at the metal-binding site and that proximity and favorable orientation are probably the most important parameters in determining the cleavage sites.

Purpose: This prospective study was conducted to compare the single-socket and the bi-socket anterior cruciate ligament (ACL) reconstruction techniques in terms of outcome., Type of Study: Nonrandomized control trial., Methods: There were 160 consecutive patients with unilateral chronic ACL insufficiency who underwent endoscopic single- or bi-socket ACL reconstruction alternately using multiple-stranded medial hamstring tendon and EndoButton (Smith & Nephew, Andover, MD) femoral fixation. All patients underwent the same postoperative rehabilitation protocol. Of them, 106 patients (57 single, 49 bi) were available for 2-year follow-up., Results: According to the IKDC Knee Ligament Evaluation Form, 23 patients (40%) of the single-socket group were subjectively graded as normal, 30 (53%) as nearly normal, and 4 (7%) as abnormal. Twenty-six patients (53%) of the bi-socket group were graded as normal, 21 (43%) as nearly normal, and 2 (4%) as abnormal (P =.19). The mean side-to-side anterior laxity difference (KT-1000 manual maximum force) was 0.9 +/- 1.8 mm for the single-socket group and 0.7 +/- 1.2 mm for the bi-socket group (P =.44). Fifty-three of 57 patients (93%) in the single-socket group and all patients in the bi-socket group showed anterior laxity differences of +/-3 mm or less (P =.12). There were no differences in thigh muscle strength between the groups., Conclusions: Both single- and bi-socket ACL reconstruction using autogenous multiple-stranded hamstring tendons with EndoButton fixation provided satisfactory anterior stability, and there were no statistically significant differences in subjective results or measured restored stability between the 2 groups.

Human meniscus cells from 47 surgically excised menisci were grown in primary culture. Cell proliferation and morphologic features were evaluated in three different culture media. Human meniscus cells showed three distinguishable cell types in monolayer culture: elongated fibroblastlike cells, polygonal cells, and small round chondrocytelike cells. These cells proliferated in Dulbecco's modified Eagle's medium, but by Day 7, elongated fibroblastlike cells became predominant. Cells did not proliferate in Ham's nutrient mixture-F-12. In a mixture of Ham's nutrient mixture-F-12 and Dulbecco's modified Eagle's medium, cells proliferated, maintaining their morphologic features and their ability to express messenger ribonucleic acids for aggrecan and Types I, II, and III collagen. Hyaluronan enhanced cellular proliferation without altering morphologic features or chondroitin sulfate production. Cultured human meniscus cells attached to a porous collagen sponge after cell seeding. Gene transfer was successful and an introduced gene was expressed by the cells, indicating that human meniscus cells can undergo gene manipulation. The finding that cells collected from small surgical specimens of human meniscus could be cultured, propagated, and seeded onto a collagen scaffold holds promise for the development of a cell-based, tissue engineered collagen meniscus.

Purpose: Although anterior cruciate ligament (ACL) reconstruction with multistrand autogenous hamstring tendons has been widely performed using a single femoral socket (SS), it is currently advocated to individually reconstruct 2 bundles of the ACL using 2 femoral sockets (TS). However, the difference in biomechanical characteristics between them is unknown. The objective of this study was to clarify their biomechanical differences., Type of Study: This is a cross-over trial using cadaveric knees., Methods: Seven intact human cadaveric knees were mounted in a robotic simulator developed in our laboratory. By applying anterior and posterior tibial load up to +/- 100 N at 0 degrees, 15 degrees, 30 degrees, 60 degrees, and 90 degrees of flexion, tibial displacement and load were recorded. After cutting the ACL, the knees underwent ACL reconstruction using TS, followed by that using SS, with 44 or 88 N of initial grafts tension at 20 degrees of flexion. The above-mentioned tests were performed on each reconstructed knee., Results: The tibial displacement in the TS technique was significantly smaller than that in the SS at smaller flexion angles in response to anterior and posterior tibial load of +/- 100 N, and the in situ force in the former was significantly greater than that in the latter at smaller flexion angles. Furthermore, in the TS technique, the posterolateral graft acted dominantly in extension, while the anteromedial graft mainly resisted against anterior tibial load in flexion. However, in the SS technique, the anteriorly located graft functioned more predominantly than the posteriorly located graft at all flexion angles., Conclusions: The ACL reconstruction via TS using quadrupled hamstring tendons provides better anterior-posterior stability compared with the conventional reconstruction using a single socket.

A total of 30 patients who underwent endoscopic reconstruction of the anterior cruciate ligament using quadrupled hamstring tendons, through a single drill hole in the femur, had MRI 24 to 28 months after operation. In 18 patients the scans revealed that both the anterior and posterior portions of the graft ran in parallel from the inside of the femoral to the tibial tunnel. In 12, the posterior bundle had moved anteriorly and the anterior bundle could not be identified at the anterodistal border of the femoral tunnel. The mean difference in the anterior laxity, when compared with the contralateral knee, was 2.0+/-1.7 mm and 4.3+/-2.8 mm for the two types, respectively. Damage to the anterior bundle may occur when using the endoscopic technique because of biomechanical disadvantages, including concentration of loading and repetitive bending stress in the anterior bundle at the opening of the femoral tunnel.

With a conventional method of RNA extraction using an acid guanidinium thiocyanate-water-saturated phenol-chloroform mixture, extraction efficiency of extractable RNA to total RNA (extractable RNA+ residual RNA) in rice leaves at various ages was 54-69%. With a new method, an improvement of the above, using benzyl chloride instead of water-saturated phenol together with further maceration with a small amount of quartz sand, the efficiency was increased to 81-95%. When RNA fractions obtained with the improved method were subjected to agarose gel electrophoresis, intact bands of 25 S and 17 S rRNAs were detected. With a DNA probe for rice rbcS, only a single band was observed on the blotted membrane. These results indicate that the improved extraction method of RNA with benzyl chloride is useful for quantitative and qualitative analysis of RNA in plant tissues such as stiff leaves of rice.

According to the results of to a survey conducted by the Japanese Society of Nuclear Medicine Technology in 1995, the mean monthly exposure dose to the trunk of nuclear medicine technicians was less than 0.2 mSv at more than 75% of the institutions, whereas the exposure dose to the fingers exceeded 0.5 mSv at 30% of the institutions. Many recent radiopharmaceuticals are being supplied as the syringe type, and while the syringe is surrounded by a tungsten or lead-glass shield, there is no shielding of the syringe needle or the plungers. The plastic plunger provides little shielding effect, and even when a tungsten plunger is used, calculating back from the leakage rate, the shielding effect for 99mTc is approximately 75%. We therefore trial-manufactured a plunger devised in such a manner as to considerably reduce exposure of the fingers and evaluated its shielding effect from leakage rate obtained with a dual-detector scintillation camera when the radionuclides 99mTc, 67Ga, 201Tl, and 123I were used. Its performance was satisfactory, with shielding effects (99mTc, 99%; 67Ga, 95%, 201Tl, 95%; 123I, 73%) about the same or better than back-calculated from the leakage rates.

The opportunities to use the syringe-type radiopharmaceuticals being supplied by manufacturers to conduct examinations in the ordinary clinical practice of nuclear medicine have recently increased, and the radiation doses to the fingers of those performing the examinations has been reduced. However, not much is known about the shielding effect of the syringe-type syringe shields and plungers. In order to evaluate their shielding effect, we devised a method in which leakage rates are calculated from the counts of the image data acquired with a 2-detector scintillation camera and compared with the values and graphs. The values made it possible to determine and compare single-direction leakage rates, whereas the graphs made it possible to determine and compare 360 degrees-direction leakage rates. This method is convenient, enables satisfactory result to be obtained in any institution, and appears to make it easy to understand the shielding effect. The validity of this method was confirmed in a comparative assessment with a survey-meter.

A newly synthesized reagent, KNK437, has been found specifically to inhibit the synthesis of heat shock proteins in vitro. In this study, we investigated the effects of KNK437 on the synthesis of heat shock proteins and the induction of thermotolerance in transplantable tumors in vivo. SCC VII cells were grown in vivo and transplanted into C3H/He mice. The concentrations of KNK437 in the tumors and the sera of the mice were examined by high-performance liquid chromatography. Hsp72 synthesis was examined by Western immunoblot analysis. The response to hyperthermia was evaluated in terms of the delay in tumor growth. KNK437 had low toxicity in vivo. The concentration of KNK437 in the tumors gradually increased and reached a peak 6 h after i.p. injection. Hsp72 were synthesized 8 h after hyperthermia at 44 degrees C for 10 min, and their synthesis was inhibited by administration of KNK437 6 h before hyperthermia. At a concentration of 200 mg/kg, KNK437 alone showed no antitumor effects and did not increase the thermosensitivity of nontolerant tumors. The same dose of KNK437 enhanced the antitumor effects of fractionated heat treatment at 44 degrees C in a synergistic manner. This study strongly suggests the inhibition of thermotolerance via the inhibition of HSP72 in vivo. The inhibition of thermotolerance by KNK437 may help to improve the efficacy of clinical fractionated hyperthermia.

Ribulose-1,5-bisphosphate carboxylase (Rubisco) efficiency for CO2-saturated photosynthesis was examined in leaves of rice (Oryza sativa L.). The amount of Rubisco in a leaf was calculated to be 30-55% in excess for the light-saturated rate of photosynthesis at 100 Pa CO2. Long-term exposure to CO2 enrichment decreased the amount of Rubisco protein. However, N was not reallocated from decreased Rubisco to other components limiting photosynthesis, and the decrease in Rubisco was simply due to a decrease in total leaf-N content by CO2 enrichment. Thus, rice plants did not optimize N allocation into Rubisco at elevated CO2. Transgenic rice plants with decreased Rubisco were obtained by transformation with the rbcS antisense gene. The transformant with 65% wild-type Rubisco was selected as a plant with optimal Rubisco content for CO2-saturated photosynthesis at the level of a single leaf. This selected transgenic plant had 20% lower rates of photosynthesis at normal CO2 (36 Pa), but 5-15% higher rates of photosynthesis at elevated CO2 (100 Pa) for a given leaf N content. However, such transgenic plants did not necessarily show greater production of biomass even under conditions of CO2 enrichment. Although they had a higher N-use efficiency for plant growth under such conditions during the middle stage of growth, the growth rate was lower during the early stage of growth. Thus, improvement of N-use efficiency by a single leaf did not necessarily lead to greater production of biomass by the whole plant.

The inhibitory effects of vitamins A and K toward P4501A1-dependent 7-ethoxycoumarin O-deethylation were examined in the reconstituted system containing the microsomal fraction prepared from the recombinant Saccharomyces cerevisiae cells producing rat P4501A1 and yeast NADPH-P450 reductase. On vitamins A, all-trans-retinol, all-trans-retinal, all-trans-retinoic acid and retinol-palmitate showed competitive inhibition with K(i) values of 0.068, 0.079, 2.6 and 2.0 microM, respectively. Judging from the K(i) values, the inhibitory effects of those vitamins A appear to have physiological significance on the basis of their contents in liver, lung and kidney. On vitamins K, vitamin K(1) showed competitive inhibition with K(i) value of 24 microM, while vitamin K(2) showed noncompetitive inhibition with K(i) value of 60 microM. Judging from these K(i) values together with the contents of these vitamins K in liver, the inhibitory effects of the vitamins K are not as significant as those of vitamins A. These results suggest that the ingestion of enough amounts of vitamins A from foods might lead to the inhibition of the activity of P4501A1 which is known to be induced by smoking, drugs such as omeprazole and lansoprazole, and environmental pollutants like dioxins., (Copyright 1999 Academic Press.)

The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) is degraded into an N-terminal side fragment of 37 kDa and a C-terminal side fragment of 16 kDa by the hydroxyl radical in the lysates of chloroplasts in light (H. Ishida et al. 1997, Plant Cell Physiol 38: 471-479). In the present study, we demonstrate that this fragmentation of the LSU also occurs in the same manner in intact chloroplasts, and discuss the mechanisms of the fragmentation. The fragmentation of the LSU was observed when intact chloroplasts from wheat leaves were incubated under illumination in the presence of KCN or NaN3, which is a potent inhibitor of active oxygenscavenging enzyme(s). The properties, such as molecular masses and cross-reactivities against the site-specific anti-LSU antibodies, of the fragments found in the chloroplasts were the same as those found in the lysates. These results indicate that, as in the lysates, the fragmentation of the LSU in the intact chloroplasts was also caused by the hydroxyl radical generated in light. The fragmentation of the LSU was completely inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), and only partially inhibited by methyl viologen in the lysates. The addition of hydrogen peroxide to the lysates stimulated LSU fragmentation in light, but did not induce any fragmentation in darkness. Thus, we conclude that both production of hydrogen peroxide and generation of the reducing power at thylakoid membranes in light are essential requirements for fragmentation of the LSU.

The release of norepinephrine (NE) in the ventral hippocampus was studied in rats with microdialysis method. The basal release of NE with perfusion of normal artificial cerebrospinal fluid (ACSF) was 1.58 +/- 0.37 x 20 microliters-1 sample. The NE concentration increased significantly with perfusion of high potassium (60 mM) ACSF indicating that depolarization-induced release was up to 5 times higher than the basic level. Ketamine (20 mg.kg-1 and 80 mg.kg-1 im) significantly inhibited the depolarization-induced increase of NE, but did not affect the basal release. Neither 5 mg.kg-1 im of ketamine nor MK-801 had any effect on the basic or the depolarization-induced release. These results suggest that the inhibitory effect of ketamine on the depolarization-induced NE release was not due to the NMDA channel blocking properties of ketamine.

Male (NZW x BXSB)F1 mice spontaneously develop a disease which closely resembles human systemic autoimmune disease, involving idiopathic thrombocytopenic purpura and glomerulonephritis. We investigated whether autoimmune thrombocytopenia in the mice responded to deoxyspergualin, as immunosuppressant. Deoxyspergualin completely prevented the development of thrombocytopenia and suppressed the increase in circulating autoantibodies against platelets. This agent also ameliorated lupus nephritis. These findings suggest that deoxyspergualin may be effective in the prevention of idiopathic thrombocytopenic purpura.

Female MRL/lpr mice develop lesions closely resembling human systemic lupus, and therefore can serve as models in order to examine the efficacy of immunosuppressive agents. The present study was designed to evaluate the efficacy of the combination of deoxyspergualin with prednisolone compared with each alone in 13-week-old female MRL/lpr mice. After the onset of lymphadenopathy, splenomegaly, and the elevation of plasma autoantibodies, deoxyspergualin alone or prednisolone alone was effective. An immunosuppressive regimen of deoxyspergualin combined with prednisolone was demonstrated to be superior to each single therapy concerning the amelioration of advanced disease in the MRL/lpr mice without increasing toxicity.

Nitrogen accumulation in the apical spikelets on the primary branches of the main stem of rice plants have been studied during the ripening process (0-35 d after flowering). The level of NADH-dependent glutamate synthase (GOGAT) protein and activity increased 4- and 6-fold, respectively, in the first 15 d after flowering. Maximum levels of NADH-GOGAT were found at that time when the spikelets had just begun to increase in dry weight and to accumulate storage proteins. Subsequently, both the level of NADH-GOGAT protein and its activity in spikelets declined rapidly. Although changes in ferredoxin (Fd)-dependent GOGAT paralleled changes in NADH-GOGAT, the relative abundance of NADH-GOGAT protein in the spikelets was about 3 times higher than that of Fd-GOGAT from 5 to 15 d after flowering. When the chaff (lemma and palea) was separated from the spikelets 10 d after the flowering, 16% of the NADH-GOGAT protein was found in the chaff and 84% in the young grain tissues (endosperm, testae, aleurone tissues, and embryo). On the other hand, Fd-GOGAT protein was distributed 52% in the chaff and 48% in the young grain tissues in spikelets of the same age. Activity of NADP-isocitrate dehydrogenase, which may generate the 2-oxoglutarate required for the GOGAT reactions, was much higher than that of total GOGAT activities on a spikelet basis during the ripening process. These results suggest that in rice plants NADH-GOGAT is responsible for the synthesis of glutamate from the glutamine that is transported from senescing tissues to the spikelets.

The amounts of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), total chlorophyll (Chl), and total leaf nitrogen were measured in fully expanded, young leaves of wheat (Triticum aestivum L.), rice (Oryza sativa L.), spinach (Spinacia oleracea L.), bean (Phaseolus vulgaris L.), and pea (Pisum sativum L.). In addition, the activities of whole-chain electron transport and carbonic anhydrase were measured. All plants were grown hydroponically at different nitrogen concentrations. Although a greater than proportional increase in Rubisco content relative to leaf nitrogen content and Chl was found with increasing nitrogen supply for rice, spinach, bean, and pea, the ratio of Rubisco to total leaf nitrogen or Chl in wheat was essentially independent of nitrogen treatment. In addition, the ratio of Rubisco to electron transport activities remained constant only in wheat. Nevertheless, gas-exchange analysis showed that the in vivo balance between the capacities of Rubisco and electron transport in wheat, rice, and spinach remained almost constant, irrespective of nitrogen treatment. The in vitro carbonic anhydrase activity in wheat was very low and strongly responsive to increasing nitrogen content. Such a response was not found for the other C(3) plants examined, which had 10- to 30-fold higher carbonic anhydrase activity than wheat at any leaf-nitrogen content. These distinctive responses of carbonic anhydrase activity in wheat were discussed in relation to CO(2)-transfer resistance and the in vivo balance between the capacities of Rubisco and electron transport.

To further explore the function of NADH-dependent glutamate synthase (GOGAT), the tissue distribution of NADH-GOGAT protein and activity was investigated in rice (Oryza sativa L.) leaves. The distributions of ferredoxin (Fd)-dependent GOGAT, plastidic glutamine synthetase, and cytosolic glutamine synthetase proteins were also determined in the same tissues. High levels of NADH-GOGAT protein (33.1 mug protein/g fresh weight) and activity were detected in the 10th leaf blade before emergence. The unexpanded, nongreen portion of the 9th leaf blade contained more than 50% of the NADH-GOGAT protein and activity per gram fresh weight when compared with the 10th leaf. The expanding, green portion of the 9th leaf blade outside of the sheath contained a slightly lower abundance of NADH-GOGAT protein than the nongreen portion of the 9th blade on a fresh weight basis. The fully expanded leaf blades at positions lower than the 9th leaf had decreased NADH-GOGAT levels as a function of increasing age, and the oldest, 5th blade contained only 4% of the NADH-GOGAT protein compared with the youngest 10th leaf blade. Fd-GOGAT protein, on the other hand, was the major form of GOGAT in the green tissues, and the highest amount of Fd-GOGAT protein (111 mug protein/g fresh weight) was detected in the 7th leaf blade. In the nongreen 10th leaf blade, the content of Fd-GOGAT protein was approximately 7% of that found in the 7th leaf blade. In addition, the content of NADH-GOGAT protein in the 10th leaf blade was about 4 times higher than that of Fd-GOGAT protein. The content of plastidic glutamine synthetase polypeptide was also the highest in the 7th leaf blade (429 mug/g fresh weight) and lowest in nongreen blades and sheaths. On the other hand, the relative abundance of the cytosolic glutamine synthetase polypeptide was the highest in the oldest leaf blade, decreasing to 10 to 20% of that value in young, nongreen leaves. These results suggest that NADH-GOGAT is important for the synthesis of glutamate from the glutamine that is transported from senescing source tissues through the phloem in the nongreen sink tissues in rice leaves.

Tissue localizations of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), chloroplastic GS (GS2), and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in rice (Oryza sativa L.) leaf blades were investigated using a tissue-print immunoblot method with specific antibodies. The cross-sections of mature and senescent leaf blades from middle and basal regions were used for tissue printing. The anti-GS1 antibody, raised against a synthetic 17-residue peptide corresponding to the deduced N-terminal amino acid sequence of rice GS1, cross-reacted specifically with native GS1 protein, but not with GS2 after transfer onto a nitrocellulose membrane. Tissue-print immunoblots showed that the GS1 protein was located in large and small vascular bundles in all regions of the leaf blade prepared from either stage of maturity. On the other hand, GS2 and Fd-GOGAT proteins were mainly located in mesophyll cells. The intensity of the developed color on the membrane for GS1 was similar between the two leaf ages, whereas that for GS2 and Fd-GOGAT decreased during senescence. The tissue-specific localization of GS1 suggests that this GS isoform is important in the synthesis of glutamine, which is a major form of nitrogen exported from the senescing leaf in rice plants.

The present paper demonstrates the efficacy of a novel immunosuppressive agent, deoxyspergualin (DSG), in rat skin transplantation despite major histocompatible antigen differences, both by itself and in combination with cyclosporin A (CyA) and prednisolone (PD). DSG significantly prolonged the median survival time (MST) of WKAH skin on F344 rats, when given at daily doses of 1.5-12 mg/kg i.p. for 10 days starting from day one after grafting. A significant increase of the MST has been observed also in the rats orally receiving CyA at daily doses of 12.5-50 mg/kg according to the same dosing protocol. DSG was as effective as CyA in prolonging the MST. In contrast, when treatment started 4 days after grafting (at the time of the rejection crisis), DSG (6 mg/kg) was able to reverse the rejection, whereas CyA (50 mg/kg) was not. When DSG (1.5 mg/kg) and CyA (6.25 or 12.5 mg/kg) were given together from day one after grafting, the combined therapy was superior to each monotherapy. Similarly, when DSG (1.5 mg/kg) and PD (10 mg/kg) were given simultaneously from the rejection crisis, the combined therapy was demonstrated to have stronger activity than any of the monotherapies. Moreover, the skin-allografted rats could be switched successfully either from CyA to DSG treatment or from DSG to CyA treatment.

We have examined the efficacy of deoxyspergualin (DSG) in protecting splenic colony-forming cells (CFU-S) from mitomycin C (MMC)-induced damage. The main findings of the study are as follows. (1) When DSG was administered at doses of 1.5 and 3 mg/kg for 7 days before the MMC injection, the decrease of the femoral CFU-S caused by MMC was diminished on the day after the MMC injection. The optimal dose was found to be 3 mg/kg. (2) In animals receiving 3 mg/kg DSG for at least 3 days preceding the MMC injection, the femoral CFU-S was more than 200% of that in the MMC alone group one day after the MMC injection. (3) The number of femoral CFU-S in the mice which received 3 mg/kg DSG for 3 days prior to MMC was significantly restored day by day and reached 70% of normal at 5 days after the MMC injection, while it was only 13% of normal in the MMC alone group. Moreover, the prior DSG administration significantly diminished the MMC toxicity to circulating platelets. (4) DSG administration (3 mg/kg) 3 days prior to MMC did not weaken the antitumor activity against colon 26 adenocarcinoma or P388 leukemia when compared with MMC alone. These findings have shown the ability of DSG specifically to protect the animals against bone marrow toxicity caused by MMC without interfering with the antitumor activity.

Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of their corresponding mRNAs have been investigated in segments of the 13th leaf of hydroponically grown rice (Oryza sativa L.) plants during natural senescence. The leaf blade on the main stem at early (0 day), middle (15 days), and late (25 days) stages of senescence was harvested and cut into 18 or 19 segments, 2 centimeters in length from the base to the tip. The amount of GS1 polypeptide, detected with specific antibody for the GS1, was greatest near the middle of the leaf blade (segments 11-13). There was little difference in the GS1 content between corresponding leaf segments obtained at the early and middle stages of senescence. At the late senescence stage, all segments had lost some GS1 polypeptide, but more than 50% of GS1 detected at both the early and middle stages was still detectable in segments. The relative content of mRNA for GS1 in the total RNA in all segments was very low during early senescence but increased in all leaf segments during later senescence. At the late stage of senescence, GS1 mRNA in the total RNA increased about 4.2- to 4.6-fold in segments 12 to 16 in the day-25 samples compared with those in the early stage. The content of the GS2 polypeptide, as well as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein, was highest in segment 17 in the 0-day samples. During senescence, this peak became lower and broader, and finally disappeared, i.e. approximately 80% of GS2 polypeptide and Rubisco protein in segment 17 were lost by day 25. In contrast with GS2 polypeptide, the relative level of GS2 mRNA increased 1.8- to 2.9-fold in individual segments at the middle stage of senescence. Even at the late stage, the transcript signals remained slightly higher than those at the early stage in all segments. Thus, GS1 and GS2 polypeptides and corresponding mRNAs responded in a different manner within an attached rice leaf during natural senescence. The contents of GS1 and GS2 polypeptides were not simply determined by the abundance of their corresponding mRNAs in the rice leaf blades during natural senescence.

Effects of irradiance on photosynthetic characteristics were examined in senescent leaves of rice (Oryza sativa L.). Two irradiance treatments (100 and 20% natural sunlight) were imposed after the full expansion of the 13th leaf through senescence. The photosynthetic rate was measured as a function of intercellular CO(2) pressure with a gas-exchange system. The amounts of cytochrome f, coupling factor 1, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and chlorophyll were determined. The coupling factor 1 and cytochrome f contents decreased rapidly during senescence, and their rates of decrease were much faster from the 20% sunlight treatment than from the full sunlight treatment. These changes were well correlated with those in the photosynthetic rate at CO(2) pressure = 600 microbars, but not with those under the ambient air condition (350 microbars CO(2)) and 200 microbars CO(2). This suggested that the amounts of coupling factor 1 and cytochrome f from the full sunlight treatment cannot be limiting factors for the photosynthetic rate at ambient air conditions. The Rubisco content also decreased during senescence, but its decrease from the 20% sunlight treatment was appreciably retarded. However, this difference was not reflected in the photosynthetic rates at the ambient and 200 microbars CO(2). This implied that in vivo Rubisco activity may be regulated in the senescent leaves from the 20% sunlight treatment. The chlorophyll content decreased most slowly. In the 20% sunlight treatment, it remained apparently constant with a decline in chlorophyll a/b ratio. These photosynthetic characteristics of the senescent rice leaves under low irradiance were discussed in relation to acclimation of shade plants.

Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of corresponding mRNAs were determined in leaves of hydroponically grown rice (Oryza sativa) plants during natural senescence. The plants were grown in the greenhouse for 105 days at which time the thirteenth leaf was fully expanded. This was counted as zero time for senescence of the twelfth leaf. The twelfth leaf blade on the main stem was analyzed over a time period of -7 days (98 days after germination) to +42 days (147 days after germination). Total GS activity declined to less than a quarter of its initial level during the senescence for 35 days and this decline was mainly caused by a decrease in the amount of GS2 polypeptide. Immunoblotting analyses showed that contents of other chloroplastic enzymes, such as ribulose-1,5-bisphosphate carboxylase/oxygenase and Fd-glutamate synthase, declined in parallel with GS2. In contrast, the GS1 polypeptide remained constant throughout the senescence period. Translatable mRNA for GS1 increased about fourfold during the senescence for 35 days. During senescence, there was a marked decrease in content of glutamate (to about one-sixth of the zero time value); glutamate is the major form of free amino acid in rice leaves. Glutamine, the major transported amino acid, increased about threefold compared to the early phase of the harvest in the senescing rice leaf blades. These observations suggest that GS1 in senescing leaf blades is responsible for the synthesis of glutamine, which is then transferred to the growing tissues in rice plants.

The beneficial effect of deoxyspergualin (DSG, NKT-01) on lethal graft-versus-host disease in mice has been studied in a major histoincompatible donor-recipient combination. Suppression of the effector mechanisms responsible for the lethal outcome in this GVHD model is also noted. This study reveals: (1) DSG has a marked potential for treatment of lethal GVHD; (2) DSG and methotrexate in combination yield longer survival times than DSG or MTX alone; (3) long-term survivors, following DSG treatment, become stable chimeras as evidenced by cell-surface analysis of spleen cells; and (4) high activity of H-2-reactive cytotoxic T lymphocytes is detected in spleens of the mice with lethal GVHD, whereas natural killer activity is only slightly increased. DSG inhibits CTL activity not only in the induction stage but also in the advanced stage of the disease. These findings indicate that DSG might be beneficial in clinical bone marrow transplantation either alone or in combination with MTX.

The present study was designed to evaluate the therapeutic activity of a novel immunosuppressive agent, deoxyspergualin (DSG, NKT-01) in male MRL/MpJ-lpr/lpr (MRL/lpr) mice suffering advanced systemic lupus erythematosus (SLE)-like lesions. Treatment with DSG in the early phase of the disease at doses of 1.5 and 3 mg/kg strongly suppressed the development of SLE-like lesions. When DSG was administered from week 21 through 29 to MRL/lpr mice in advanced phases of the disease, a daily iv dose of 3 mg/kg (5 days/week) markedly reduced the symptoms, whereas a dose of 1.5 mg/kg did not. Moreover, DSG treatment at a dose of 3mg/kg, started at the time when the blood urea nitrogen levels were over 50 mg/deciliter, significantly prevented deterioration of the hyperuremia. Taking these findings into consideration, DSG was found to be a promising agent for curing such established autoimmune disease.

The lysate from intact chloroplasts mechanically isolated from primary leaves of 9 day old seedlings of wheat (Triticum aestivum L. var Aoba) was incubated in the pH range of 5.5 to 8.5 at 37 degrees C for 5 hours. Proteolytic activity against ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was estimated by disappearance of the large subunit of Rubisco or the appearance of its degradation products. Although the activity in lysates was weak, the products were detected by applying Western blotting. The degradation products were similar to those obtained when Rubisco was incubated with the lysate of vacuoles isolated from like leaves. Although some of the products were similar to those from vacuole lysates, many were clearly different after incubation of Rubisco with trypsin, V-8 protease, or reactive oxygen (hydroxy radical). Lysates of chloroplasts, pretreated with thermolysin at 4 degrees C for 30 minutes, had no proteolytic activity against Rubisco after incubation at 37 degrees C for 5 hours. These results show that the proteolytic activity against Rubisco found in lysates of our mechanically isolated chloroplasts was mostly due to the contamination of vacuolar proteases adhering to the outer envelope of the chloroplasts during their isolation.

Changes in photosynthesis and the ribulose 1,5-bisphosphate (RuBP) carboxylase level were examined in the 12th leaf blades of rice (Oryza sativa L.) grown under different N levels. Photosynthesis was determined using an open infrared gas analysis system. The level of RuBP carboxylase was measured by rocket immunoelectrophoresis. These changes were followed with respect to changes in the activities of RuBP carboxylase, ribulose 5-phosphate kinase, NADP-glyceraldehyde 3-phosphate dehydrogenase, and 3-phosphoglyceric acid kinase.RuBP carboxylase activity was highly correlated with the net rate of photosynthesis (r = 0.968). Although high correlations between the activities of other enzymes and photosynthesis were also found, the activity per leaf of RuBP carboxylase was much lower than those of other enzymes throughout the leaf life. The specific activity of RuBP carboxylase on a milligram of the enzyme protein basis remained fairly constant (1.16 +/- 0.07 micromoles of CO(2) per minute per milligram at 25 degrees C) throughout the experimental period.Kinetic parameters related to CO(2) fixation were examined using the purified carboxylase. The K(m)(CO(2)) and V(max) values were 12 micromolar and 1.45 micromoles of CO(2) per minute per milligram, respectively (pH 8.2 and 25 degrees C). The in vitro specific activity calculated at the atomospheric CO(2) level from the parameters was comparable to the in situ true photosynthetic rate per milligram of the carboxylase throughout the leaf life.The results indicated that the level of RuBP carboxylase protein can be a limiting factor in photosynthesis throughout the life span of the leaf.

The enzymic properties of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase purified from rice (Oryza sativa L.) leaves were studied. Rice RuBPcarboxylase, activated by preincubation with CO(2) and Mg(2+) like other higher plant carboxylases, had an activation equilibrium constant (K(c)K(Mg)) of 1.90 x 10(5) to 2.41 x 10(5) micromolar(2) (pH 8.2 and 25 degrees C). Kinetic parameters of carboxylation and oxygenation catalyzed by the completely activated enzyme were examined at 25 degrees C and the respective optimal pHs. The K(m)(CO(2)), K(m)(RuBP), and V(max) values for carboxylation were 8 micromolar, 31 micromolar, and 1.79 units milligram(-1), respectively. The K(m)(O(2)), K(m)(RuBP), and V(max) values for oxygenation were 370 micromolar, 29 micromolar, and 0.60 units milligram(-1), respectively.Comparison of rice leaf RuBP carboxylase with other C(3) plant carboxylases showed that it had a relatively high affinity for CO(2) but the lowest catalytic turnover number (V(max)) among the species examined.

Changes in gas-exchange rates during the life span of the leaves of rice (Oryza sativa L.) were analyzed quantitatively by measuring changes in the carboxylation/oxygenation and regeneration of ribulose 1,5-bisphosphate (RuBP) at photon fluence rates of 2000 (saturating) and 500 (subsaturating) μmol quanta·m(-2)·s(-1) under ambient air conditions. The RuBP levels were always higher than the active-site concentrations of RuBP carboxylase (EC 4.1.1.39), irrespective of the irradiance supplied. Analysis of the CO2-assimilation rate as a function of intercellular CO2 concentration indicated that RuBP regeneration does not limit CO2 assimilation. The estimated RuBP-carboxylase/oxygenase activity in vivo was linearly correlated to the rate of CO2 assimilation at each level of irradiance. This enzyme activity was just enough to account for the rate of CO2 assimilation at the saturating irradiance and was 35% more than the rate of CO2 assimilation at the subsaturating irradiance. Analysis of the assimilation rate at subsaturating irradiance as a function of intercellular CO2 concentration indicated that a limitation caused by enzyme activation comes into play. The results indicate that the rate of CO2 assimilation in rice leaves under ambient air conditions is limited during their entire life span by the RuBP-carboxylation/oxygenation capacity.

The kinetic parameters of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39) in wheat (Triticum aestivum L.) and rice (Oryza sativa L.) were determined by rapidly assaying the leaf extracts. The respective K m and V max values for carboxylase and oxygenase activities were significantly higher for wheat than for rice. In particular, the differences in the V max values between the two species were greater. When the net activity of CO2 exchange was calculated at the physiological CO2-O2 concentration from these kinetic parameters, it was 22% greater in wheat than in rice. This difference in the in-vitro RuBP-carboxylase/oxygenase activity between the two species reflected a difference in the CO2-assimilation rate per unit of RuBP-carboxylase protein. However, there was no apparent difference in the CO2-assimilation rate for a given leaf-nitrogen content between the two species. When the RuBP-carboxylase/oxygenase activity was estimated at the intercellular CO2 pressure from the enzyme content and kinetic parameters, these estimated enzyme activities in wheat and rice were similar to each other for the same rate of CO2 assimilation. These results indicate that the difference in the kinetic parameters of RuBP carboxylase between the two species was offset by the differences in RuBP-carboxylase content and conductance for a given leaf-nitrogen content.